Microbial proteases are among the most important hydrolytic enzymes and find applications in various industrial sectors, such as detergents, food, leather, pharmaceuticals, diagnostics, waste management and silver recovery. Microbial extracellular proteases account for a major part of the total worldwide industrial enzyme sales (Cherry and Fidantsef, 2003). Approximately 90% of the commercial proteases are detergent enzymes (Gupta et al., 2002). The commercial detergent preparations currently in use comprise the naturally occurring alkaline serine proteases (EC 3.4.21) of the subtilisin family or subtilisins (EC 3.4.21.62), originating from Bacillus species, or are recombinant protease preparations thereof (Maurer, 2004).
Examples of commercial proteases are such as Subtilisin Carlsberg (Alcalase®), Subtilisin 309 (Savinase®), Subtilisin 147 (Esperase®), Kannase®, Everlase®, Ovozyme®, and the cold-wash protease Polarzyme® (Novozymes A/S, DK), Purafect®, Purafect® Ox, Purafect® Prime and Properase® (Genencor Int., Inc., USA), and the BLAP S and X series (Henkel, Del.).
Several alkaline serine proteases and genes encoding these enzymes have also been isolated from eukaryotic organisms, including yeast and filamentous fungi. U.S. Pat. No. 3,652,399 and EP 519229 (Takeda Chemical Industries, Ltd., JP) disclose an alkaline protease from the genus Fusarium (asexual state, teleomorph) or Gibberella, (sexual state, anamorph) particularly from Fusarium sp. S-19-5 (ATCC 20192, IFO 8884), F. oxysporum f sp. lini (IFO 5880) or G. saubinetti (ATCC 20193, IF06608), useful in the formulation of detergent and other cleanser compositions. WO1994025583 (NovoNordisk A/S, DK) discloses an active trypsin-like protease enzyme derivable from a Fusarium species, in particular a strain of F. oxysporum (DSM 2672), and the DNA sequence encoding the same. The amino acid and nucleotide sequences of the serine proteases from F. equiseti and F. acuminatum have been disclosed in WO 2010125174 and WO 2010125175, respectively (AB Enzymes Oy, FI). Also, alkaline proteases from fungal species such as Tritirachium and Conidiobolus have been reported (reviewed in Anwar and Saleemuddin 1998)
The major problem in the use of proteases in liquid detergents is their instability. In liquid detergents enzymes are in direct contact with water and chatropic agents like anionic surfactants and complexing agents, which can lead to irreversible denaturation. Proteases degrade proteins including other enzymes in detergent formulations and themselves. The auto-proteolysis is enhanced by surfactants and heat. Thus, the stability of liquid detergents containing protease represents a major challenge for product development (Maurer, 2010).
Various methods have been used for improving the stability of industrial serine proteases. WO 92/03529 (NovoNordisk A/S, DK), US 2009/096916 (Genencor Int. Inc., US) and WO 2007/145963 (Procter & Gamble Co., US) disclose the use of a reversible protease inhibitor of a peptide or protein type. Liquid detergent compositions comprising proteases often include protease inhibitors such as boric acid with or without polyols to inhibit the activity of proteases. One example of such inhibitors is 4-formyl phenyl boronic acid (4-FPBA) disclosed in US 2010/0120649 (Novozymes A/S, DK). The stability of proteases has also been improved by using a combination of halide salts with polyols (WO 02/08398, Genencor Int. Inc., US). EP 0352244A2 (NovoNordisk A/S, DK) suggests improving the stability of Bacillus derived enzymes using amphoteric compounds, such as surfactants.
Based on the information derived from the crystal structures and sequence similarity comparisons between homologous proteins, variants with improved stability and/or improved performance may be designed. Variants of the natural serine proteases with improved catalytic efficiency and/or better stability towards temperature, oxidizing agents and different washing conditions, as well as improved storage stability in liquid detergents have been developed through site-directed and/or random mutagenesis.
Thermomycolin EC 3.4.21.65, isolated as an extracellular alkaline endopeptidase, is produced by a thermophilic fungus Malbranchea pulcella var. sulfurea. Thermomycolin is described as a 325 residue, single-chain protein. It has the active-site sequence -Leu-Ser-(Gly)-Thr-Ser*-Met-, which is typical for a member of the subtilisin family. Thermomycolin possesses one disulfide bridge, which is exceptional. Thermomycolin is not as thermostable as the extracellular serine proteinases of thermophilic bacteria, but it is more stable than most fungal proteinases (Gaucher and Stevenson, 2004). According to Ong and Gaucher (1975) the thermal inactivation of thermomycolin occurs at 73° C. in the presence of 10 mM Ca2+. Thermomycolin hydrolyses casein on broad pH range. The optimum pH for hydrolysis of casein is about 8.5.
Abu-Shady et al. (2001) disclose properties of a protease from Malbranchea sulfurea that is a local isolate from soil samples collected from butcheries in Egypt and cultured to obtain a protease enzyme. This publication describes the relative activity of M. sulphurea protease in the presence of certain detergents at low concentrations (0.7%) at 30-90° C. using preincubation time of 15-60 min, i.e. at conditions resembling washing conditions. However, it does not give any indication of the stain removal performance or the storage stability of this protease in detergent itself, which are essential properties for the suitability for use of a protease in detergent formulations. The publication also describes the temperature and pH profiles of a partially purified protease. The optimum temperature of the protease is at 50° C. and optimum pH at 9.0.
Despite the fact that numerous patent publications, reviews and articles have been published, disclosing fungal serine proteases from various microorganisms, for example, the low temperature alkaline proteases from actinomycete (Nocardiopsis dassonvillei) and fungal (Paecilomyces marquandii) microorganisms, e.g. in EP 0290567 and EP 0290569 (Novo Nordisk A/S, DK), there is still a great need for new proteases, which are suitable for and effective in modifying, degrading and removing proteinaceous materials of different stains, particularly at low or moderate temperature ranges and which are stable in the presence of detergents with highly varying properties. Due to the autocatalytic property of serine proteases, the stability during storage is also very important.
It is also desirable that the serine protease can be produced in high amounts, and can be cost-effectively down-stream processed, by easy separation from fermentation broth and mycelia.